It has recently been found that a protein previously implicated in the Na+-dependent transport of inorganic phosphate also transports glutamate into synaptic vesicles for exocytotic release. In addition, vesicular glutamate transporter 1 (VGLUT1) appears to have a conductance for chloride. However, current methods have limited our understanding of the VGLUTs. To understand the multiple activities attributed to them, I now propose to mislocalize VGLUT1 at the plasma membrane of Xenopus oocytes. Expression at the cell surface will enable me to characterize its function using radiotracer flux assays, pH imaging and electrophysiology. With these assays, I will assess the various functions attributed to VGLUT1, including the Na+-dependent uptake of Pi, the potential for non-vesicular glutamate release at resting membrane potential, the ionic dependence of currents, and the relationship of the observed chloride conductance to the chloride dependence of glutamate flux, all of which have important implications for VGLUT function at the nerve terminal. In addition, the development of better assays will greatly facilitate all future work on the regulation of VGLUTs and their role in excitatory neurotransmission.